Friday, 2 June 2017

T 1818/12 - On evidence, sufficiency, and effect

This pencil cracked under the pressure

The main claim in this opposition appeal concerns
an E. coli host cell sample subjected to non-lysing pressure. According to the opponent
the examples in the patent are the only known cell systems that do not lyse under
the conditions of the claim, or that give the effect of increased yield. As a
result, the claim is neither workable nor inventive over the entire scope
claimed.

The Board has problems with the evidentiary value of the documents cited by the opponent. Furthermore, that there exists an embodiment falling under the scope of the claim that does not show the effect is in itself not enough to deny inventive step.

Summary of Facts and Submissions

(...)

VI. Claim 1 of the set of claims found allowable in the decision under appeal (main request) reads as follows:

"1. A method for the manufacture of recombinant antibody molecules comprising culturing an E. coli host cell sample transformed with an expression vector encoding a recombinant antibody molecule that is expressed in the periplasm of the host cell and subjecting said host cell sample to a heat treatment step, characterised in that said sample is subjected to a non-lysing pressure treatment step between 1000 psi

(68.9 bar) and 4000 psi (275.8 bar) before being subjected to an increase in temperature within the range of 30°C to 70°C for a period of up to 24 hours".

(...)

Reasons for the Decision

1. The oral proceedings were held in the absence of the appellant, in accordance with Rule 115(2) EPC and Article 15(3) RPBA. Accordingly, the appellant is treated as relying on its written case.

Main Request - Claims 1 to 5

Disclosure of the invention - Article 100(b) and Article 83 EPC

2. The ground for opposition Article 100(b) EPC requires that the European patent discloses the invention in a manner sufficiently clear and complete for it to be carried out by a person skilled in the art. According to the case law of the boards of appeal, the subject-matter of a patent is sufficiently disclosed if the skilled person is able to obtain substantially all embodiments falling within the ambit of the claims. Moreover, it must be possible to reproduce the invention on the basis of the patent without any inventive effort and undue burden (see Case Law of the Boards of Appeal of the European Patent Office, 8th edition, II.C. 4.1, 4.4 and 5.6)

3. The appellant argued that finding E. coli strains suitable for use in the claimed method, other than the strain W3110, was an undue burden for the skilled person. Document D6, provided evidence of cell lysis under the pressure and temperature conditions of the claim. Similarly, Annexes 1 and A were submitted as additional evidence to show, inter alia, that due to cell lysis under conditions of the claim, the skilled person would not be able to carry out the invention other than by using the strain exemplified in the patent.

4. Document D6 concerns "the high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin" (see abstract). None of the findings on the lysis of E. coli strains under homogeniser pressure (see page 368, right column, penultimate paragraph and Figure 7) relate to periplasmic expression of a protein. It was concluded that "the overexpression of a foreign protein leads to weakening of the cell wall" (Id, paragraph 1). However, in the case of periplasmic expression, the foreign protein would be expressed outside of the cell wall. In view of this, the board considers that there is no evidence in document D6 that the skilled person would encounter any difficultly in identifying strains of E. coli periplasmically expressing recombinant proteins that do not lyse under conditions of the claim.

4.1 Thus, document D6 does not provide evidence that the skilled person cannot carry out the invention as claimed without undue burden.

5. In relation to the evidence provided in Annexes 1 and A, the established case law of the boards is that there are no firm rules according to which types of evidence are, or are not, convincing. Each piece of evidence is given an appropriate weighting according to its probative value on a case-by-case basis (see Case Law of the Boards of Appeal of the European Patent Office, 8th edition, III.G.4.2).

6. In the case at hand, the board has noted a number of issues that arise when considering the Annexes. These include the following:

6.1 Neither Annex 1 nor Annex A has an identifiable author. In the case law of the boards "an unsigned statement by an unknown and unnamed person should in principle be given minimal weight" (see Id., 4.2.1). Moreover, the level of skill of the person who carried out the experiments is unknown.

6.2 It is not clear if, in the experiments reported in the Annexes, the expression of the antibody was to the periplasm. The board notes that expression of a protein to the periplasm in E. coli is generally governed by the presence of an appropriate signal sequence. Neither Annex nor any of the appellant's written submissions provide any details about the expression vector used.

6.3 The information presented in Annex 1 is, in part, inconsistent with that presented in Annex A. For instance Annex 1 (Abb. 4) and Annex A (Fig. 1b) relate to the same experiment and both show the amount of product (antibody) recovered in mass per unit volume. The yield obtained for induced and auto-induced systems at 70 and 200 bar shown in Abb. 4 of Annex 1 and Fig. 1b of Annex A is shown below;

Annex 1 (Abb.4) Annex A (Fig. 1b)

70 bar

Auto-induced ca. 50 mg/l 0 mg/l

Induced ca. 250 mg/l 1050 mg/l

200 bar

Auto-induced ca. 230 mg/l 0 mg/l

Induced ca. 420 mg/l 1200 mg/l

6.4 The fact that there is no correspondence between the yield achieved for the induced and the auto-induced systems between the two sets of results, although these relate to the same experiment, leads to the conclusion the there must be an error, either in the reporting of the results or in the experiments themselves.

7. All of the above factors lead the board to the conclusion that the evidence in both Annex 1 and A is of a nature that it cannot convincingly demonstrate that the skilled person would face an undue burden in carrying out the invention as claimed without undue burden.

8. The board is therefore satisfied that the requirements of Article 83 EPC are met for the subject-matter of the claims.

Inventive step - Article 56 EPC

9. To assess whether or not a claimed invention meets the requirements of Article 56 EPC, the board applies the "problem and solution" approach, long established in the case law of the boards of appeal (see Case Law of the Boards of Appeal of the European Patent Office, 8th edition, I.D.2).

The closest prior art

10. Document D3 discloses, inter alia, methods for the manufacture of recombinant antibodies from the periplasm of E. coli strain W3110 and retrieval of folded and soluble material by applying heat treatment (see column 13, line 64 to column 14, lines 1 to 19). This document was seen as representing the most relevant state of the art for assessing inventive step by the opposition division and both parties. The board has no reasons to depart from this assessment.

The technical problem and its solution

11. The method of claim 1 differs from that disclosed in document D3 in the inclusion of a step of subjecting the E. coli host cell sample to a non-lysing pressure treatment. According to the patent, the technical effect of this is "that non-lysing treatment in combination with heat treatment, brings an increase in the yield of functional antibody at the primary extraction stage of up to 50%; i.e. the yield of functional antibody is increased above that of heat treatment alone" (see paragraph [007]).

12. The appellant, on the basis of evidence in the patent itself, in Annexes 1 and A and in document D6, disputes that this effect is achievable over the entire scope of the claim.

13. It is established case law of the boards of appeal that "If the inventive step of a claimed invention is based on a given technical effect, [it] should, in principle, be achievable over the whole area claimed" (see Case Law of the Boards of Appeal of the European Patent Office, 8th edition 2016, ID. 9.8.3).

14. The data provided in the patent in Figures 2, 3b, 4a and Table 1 shows that the yield of antibody obtainable by carrying out the method as claimed is improved in comparison to the yield obtainable without the non-lysing pressure conditions, i.e. in comparison with a method representing the closest prior art. Figure 3(a) of the patent (a histogram showing the effect of pressure treatment on the yield of functional antibody at 60°C) shows that the yield of functional antibody at 1000 psi (319 mg/l) was slightly less than the control (atmospheric pressure; 343 mg/l). However, in the same experiment, the yield at pressures of 2000 and 4000 psi was greater than the control (460 and 921 mg/l, respectively).

15. Thus, while the patent discloses that a single set of conditions, falling within the ambit of claim 1, did not result in an improved yield, it also discloses several other working embodiments of the claimed method which did result in an improved yield. For the board, the overall evidence in the patent convincingly demonstrates that using the claimed method leads to an improved yield of high purity antibodies, achievable over substantially the whole area claimed.

16. As far as document D6 and Annexes 1 and A are concerned, the board considers that none of them provides evidence that the technical effect of improved yield of high purity antibodies cannot be achieved over the whole area claimed for the reasons given in points 4.4. to 7.7. above.

17. Hence, the technical problem can be seen as to improve the yield of a method for the production of high purity recombinant antibodies.

Obviousness

18. The question to be answered in considering obviousness is whether the person skilled in the art, seeking a solution to the above formulated problem, and starting from the closest prior art as represented by document D3, would have considered that subjecting an E. coli sample, periplasmically expressing recombinant antibody, to a non-lysing pressure treatment step at between 1000 psi and 4000 psi, before subjecting it to an increase in temperature, was obvious.

19. The appellant has argued that the solution as presented in claim 1 was obvious because it combines heat with pressure treatment which combination was derivable from the disclosure of document D3 combined with that of document D5.

20. Document D5 is a review of process-scale techniques used to disrupt host cells for the large-scale manufacture of biological products. In relation to the release of proteins from the periplasmic proteins it states "[...] chemical attack of the outer membrane allows periplasmic proteins to be released. Enzymatic methods generally involve enzymatic attack of the peptidoglycan layer in gram-negative bacteria, and of the mannoprotein and glucan components of the yeast wall" (page 499, first paragraph). More specifically it states "EDTA is clearly effective at disrupting the outer membrane, and may therefore be employed to recover periplasmic proteins" (page 501, final paragraph).

21. On the other hand, pressure treatment is mentioned on page 498 as follows: "Complete destruction of the wall in a non-specific manner is usually achieved by mechanical means. Laboratory-scale methods [...] include the French press, shaking with glass beads and sonication. At process scale, mechanical methods are restricted primarily to bead milling, high-pressure homogenization, and microfluidization...".

22. In summary, document D5 suggests chemical means for the specific release of proteins from the periplasm and discloses pressure treatments as a means for the complete destruction of the cell wall and the release of the entire intracellular content.

23. From this, the board concludes that the skilled person starting from document D3 and seeking to improve the yield of high purity antibody would not have considered including an additional pressure step before the heat treatment step, since such steps were seen as a means of totally destroying the cell wall.

24. It follows that the board holds that the subject-matter of claim 1 was not obvious to the person skilled in the art at the effective date of the patent. Dependent claims 2 to 5 relate to embodiments of claim 1. The conclusions on inventive step reached for the subject-matter of claim 1 therefore apply equally to the subject-matter of claims 2 to 5.